1. Field of the Invention
This invention relates to a surface-modified electrode and its use in a bioelectrochemical process in which electrons are transferred directly between an electrode and an electroactive biological material which is capable of accepting or donating one or more electrons. Many such materials are redox species having a reduced state in which they can accept electron(s) and an oxidised state in which they can donate electron(s). Bioelectrochemical processes of the above kind include processes of carrying out enzymatic reactions, especially for oxidising or reducing organic compounds, in which electrons are transferred from the electrode to the enzyme, to a protein with which an enzyme is complexed or to a cofactor. Other bioelectrochemical processes involve using the energy of biological materials, e.g. enzyme-producing bacteria, to donate electrons to an electrode and thereby drive a fuel cell.
The invention is concerned with a process of direct electron transfer, whereby electrons are transferred directly (mediatorlessly) to the electroactive biological material without the intervention, in that transfer, of any other redox species. Typical mediators are redox dyes and cofactors such as NAD(H) and NADP(H). The bioelectrochemical processes with which the invention is concerned involve the adsorption of the electroactive material from solution onto the surface of the electrode, whereat the electron transfer takes place directly between electrode and electroactive material.
2. Description of the Prior Art
A bioelectrochemical process of the above kind was first described in UK Pat. No. 2033428B (National Research Development Corporation). The corresponding U.S. Pat. No. 4,318,784. The patent described a process in which direct electron transfer takes place from a gold electrode to a protein exemplified by a methane mono-oxygenase enzyme derived from Methylosinus trichosporium, an enzyme complex of cytochrome p450, putidaredoxin and putidaredoxin reductase, and cytochrome c. The patent recommends use of 4,4'-bipyridyl or 1,2-bis(4-pyridyl)ethene as a promotor of the electron transfer. Subsequently, I. Taniguchi et al., J. Chem. Soc., Chem. Commun. 1032 (1982) reported 4,4'-dithiopyridine, otherwise known as bis(4-pyridyl) bisulphide, as a promotor. The process is primarily of interest to supply reducing equivalents which re-convert the oxidised form of an enzyme to its reduced form, for the enzyme catalysis of organic oxidation reactions. In other words, organic chemical reactions are driven by supplying electrical energy.
The 4,4'-bipyridyl-like promotor is not a mediator, but appears to be adsorbed onto the electrode surface to provide a suitable interface for attracting the protein. The protein most extensively studied is horse-heart (HH) cytochrome c. HH cytochrome c is known to contain residues of the amino acid lysine in a ring around the heme edge of the protein and it is believed that when HH cytochrome c forms a complex with a redox enzyme, electron transfer takes place via the heme edge. The theory is that the function of the promotor is to attract the heme edge of the cytochrome c to face the electrode. The .epsilon.-amino groups of the lysine residues near the heme edge might hydrogen-bond to the N-atom of a pyridine ring nitrogen of the 4,4'-bipyridyl molecule, the other end of which is possibly "perpendicularly" adsorbed on the electrode surface. See, for example, M. J. Eddowes and H. A. O. Hill, Faraday Discuss. Chem. Soc. 74, 331-341 (1982).
UK Pat. No. 2105750B (National Research Development Corporation) describes a bioelectrochemical process of the same kind but using a different type of electrode. Whereas in the specific description of the earlier patent gold electrode was used and the promotor was added to the electrolyte, the second patent uses an electrode incorporating a "binding species" therewithin. The binding species comprises ionic functional groups or non-ionic species giving rise to a dipole. The electroactive biological material has an oppositely charged site close to the electron transfer portion thereof, so that the biological material becomes temporarily bound to the electrode at the charged site. In the particular embodiments disclosed, the electrode is of graphite and the binding species is either an oxidised group produced by surface-oxidation of the graphite or a C.sub.10 -C.sub.30 fatty acid incorporated in the body of the graphite electrode during manufacture. The theory is that the binding species provides COO.sup.- or similar groups which, at appropriate pH, attract positively charged NH.sub.3.sup.+ groups in the lysine residues of HH cytochrome c. The binding species is therefore similar in its theorised action to a 4,4'-bipyridyl promotor, except that it is apparently attracted to lysine residues by electrostatic rather than hydrogen bonding.
Recently, P. M. Allen et al., J. Electroanal. Chem 178, 69-86 (1984), examined 54 bifunctional organic compounds to assess their ability to promote the direct electrochemistry of horse-heart cytochrome c at a gold electrode. The assessment gave rise to the conclusion that successful promotors are of the general formula X Y, where X represents a group which adsorbs or binds to the gold surface through a nitrogen, phosphorus or sulphur atom, Y represents an anionic or weakly basic functional group which binds to the positively charged cytochrome c protein ionically or by hydrogen bonding, and the wavy line joining X and Y represents a chemical linkage which can be conformationally rigid or flexible, but which must direct the binding group Y outwardly from the surface of the electrode when group X is adsorbed or bound to the electrode. These X Y promotors are termed "surface modifiers" by P. M. Allen et al., and the same terminology will be used hereinafter. Examples of these surface modifiers are 4-mercaptopyridine, 4-mercaptoaniline, 2,3-dimercaptosuccinic acid, thiodiethanoic acid, 3,3'-thiobis(propanoic acid), 2,2'-thiobis(succinic acid), 4,4 'dithiopyridine, dithiobis(ethanoic acid), 2,2'-dithiobis(succinic acid), 3-thiophenethanoic acid acid, sodium monothiophosphate, 1,2-bis(4-pyridyl)ethene, 2,5-bis(4-pyridyl)-1,3,4-thiadiazole, pyridine-4-sulphonic acid and 4-pyridylphosphonic acid. Some of these compounds required pre-activation of the surface of the electrode with 4,4,'-dithiopyridine, followed by polishing the electrode.
Negatively-charged proteins such as rubredoxin and 2[4Fe-4S] ferredoxin do not give electron transfer with a graphite electrode as described above. However, when a multivalent cation such as Mg.sup.2+ is added, they do give electron transfer, F. A. Armstrong et al., J. Amer. Chem. Soc. 106, 921-923 (1984). Clearly, the divalent Mg.sup.2+ cation bridges between the negatively charged species in the electrode and the negatively charged site in the protein.